1. Field of the Invention
The present invention relates to non-immunogenic, long acting Factor VIII or Factor IX coagulation factors. The Factor VIII or Factor IX is covalently bonded through a linker or a coupling agent to a poly(alkylene oxide).
2. Description of the Prior Art
Factor VIII and Factor IX are necessary to the human blood coagulation process. Individuals deficient in either Factor VIII or Factor IX usually require administration of the absent coagulation factor on a frequent basis for maintaining a normal blood coagulation profile. These blood proteins are obtained from the blood of donors or prepared using recombinant DNA procedures. At times, more so when obtained from pooled blood, administration of these Factors into the circulatory system can result in an adverse immunogenic response. This can precipitate antibody formation against the administered protein or an allergic response. A second major disadvantage of the currently available Factor VIII and Factor IX formulations is a relatively short half-life, requiring frequent injections.
On the other hand, polyalkylene glycols, especially polyethylene glycol and polypropylene glycol and copolymers thereof (such as Pluronics) have been linked to certain polypeptides to form polyalkylene glycol-polypeptide compounds or conjugates characterized by reduced immunogenicity and prolonged half-life. The specific polypeptides hitherto exemplified in polyalkylene oxide conjugates are distinct in their manner of biological function in comparison with Factor VIII and Factor IX.
In general, Factor VIII and Factor IX are consumed through biochemical conversions during the blood coagulation cascade. This is in contrast to substrates such as enzymes, or hormones which can conform in mating configuration with receptors.
U.S. Pat. No. 4,179,337 to Davis, et al., describes peptide enzymes and peptide hormones coupled to polyethylene glycol or polypropylene glycol of molecular weight 500 to 20,000 daltons to provide physiologically active non-immunogenic water-soluble polypeptide compositions, protected from loss of activity. Enzyme categories of oxidoreductases, tranferases, hydrolases, lyases, isomerases or lipases and the peptide hormones insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalamic releasing factors, antidiuretic hormones, thyroid stimulating hormone and prolactin are mentioned. The '337 patent discloses various coupling groups provided by linking or coupling compounds which can link a polyethylene glycol and/or polypropylene glycol to the amino nitrogen or carboxyl carbonyl moiety of the peptide.
On the other hand, Factor VIII and Factor IX are normal plasma proteins which serve as co-factors in the coagulation cascade. Both are large glycoproteins of known amino acid structure. Hemophilia A is associated with Factor VIII deficiency, hemophilia B is associated with Factor IX deficiency.
Unlike true enzymes or intact hormones which illicit their biological activity intact as pure substrates on which activity occurs or in confirmational configuration with receptors, both Factor VIII and Factor IX must undergo cleavage in order to form their active respective co-factors VIIIa and IXa. In the body, serine proteases such as thrombin or Factor Xa activate the inactive co-factors VIII and IX to VIIIa or IXa. Further, Factor VIII circulates in the blood bound to von Willebrand factor. The Factor VIII bound to the von Willebrand factor (Factor VIII:vWF) is designated as Factor VIII:C. The activated co-factor VIIIa or IXa is subsequently inactivated by another serine protease, such as Activated Protein C (APC) or by protease inhibitors.
Thus, each of Factor VIII and IX is consumed in displaying its part in hemostatic process. Indeed, Factor VIII undergoes two cleavages into smaller fragments, the first to provide the active co-factor which participates in the coagulation cascade, and the second to inactivate co-factor. But unlike some biochemical sequences, the inactivation further cleaves the activated co-factors to even smaller proteins.
In contrast, Factor IXa, a protease which is produced from Factor IX by proteolytic removal of a small glycopeptide fragment, rapidly binds antithrombin III in vivo. This complex is removed from circulation by hepatic .alpha..sub.1 antitrypsin receptors and thus specifically and rapidly cleared by the liver.
Other prior art describing the linkage of peptides to poly(alkylene oxide) to effect its immunogenicity and/or half-life, is as follows:
U.S. Pat. No. 4,495,285 is directed to the attachment of polyethylene glycol of molecular weight of 200 to 2,000 to amino acid side-chains of plasminogen activators of human origin to improve stability and half-life. All the examples thereof employ urokinase.
U.S. Pat. No. 4,609,546 utilizes as the poly(alkylene-oxide) a polyoxyethylene-polyoxypropylene co-polymer, i.e., methoxy-polyoxyethylene-polyoxypropylene glycol. The peptide can, for example, be a hormone or enzyme. Furthermore, the examples which are mentioned are human menopausal gonadotropin, human growth hormone, epidermal growth factor, nerve growth factor, colony formation stimulating factor, urokinase, plasminogen, kallikrein, interferons, interleukins, urinary trypsin inhibitor, urinary thiol protease inhibitor, placental acylsulfatase, urinary lysozyme and urinary asparaginase. The examples thereof employ urokinase, kallikrein or interferon.
U.S. Pat. No. 4,640,835 is a CIP of U.S. Pat. No. 4,495,285.
U.S. Pat. No. 4,645,741 utilizes lipases and connects them to polyalkylene glycols.
U.S. Pat. No. 4,670,417 bonds hemoglobin via amino groups to poly(alkylene oxide).
U.S. Pat. No. 4,791,192 bonds islet activating protein from biological origin via primary amino groups thereof to polyethylene glycol.
U.S. Pat. No. 4,801,451 employs enzymes while U.S. Pat. No. 4,902,502 and U.S. Pat. No. 5,037,644 employ interleukins. U.S. Pat. No. 5,006,333 employs superoxidase dismutase which is attached via amino, carboxyl or sulfhydryl groups to polyalkylene glycols.